Vaccine containing a thiol protease

ABSTRACT

Thiol proteases having Cathepsin 1 type activity are used in the formulation of vaccines for combating helminth parasites. Preferably the protease is derived from a fluke such as  Fasciola hepatica.

This application claims priority from British Application No. 9222156.3,filed Oct. 21, 1992 and British Application No. 9307028.2, filed Apr. 2,1993.

FIELD OF THE INVENTION

The invention relates to the use of a class of thiol proteases asprotective antigens against helminth parasites, namely the CathepsinL-like proteases frequently released as excretory/secretory products bysuch parasites.

DESCRIPTION OF THE RELATED ART

Each species of domestic animal can be parasitised by a number ofdifferent species of helminths, a process which usually causes disease.For example the parasitic trematode Fasciola hepatica is known to be thecause of the economically important disease fascioliasis in ruminants,such as cattle and sheep. The parasite enters the mammalian host bypenetrating the gut wall and spends approximately seven weeks feeding onand burrowing through the liver mass before migrating into the bileduct. Following infection, development of immunity in the host is poorand resistance to reinfection in already infected hosts is only partialor non-existent. Other parasitic flukes include Fasciola gigantica andDicrocoelium spp. and also Paramphistomum spp.

Problems are also caused by nematodes such as hookworms (e.g. Necator,Ancylostoma, Uncinaria and Bunostomum spp.).

Of the blood feeding nematodes the genus Haemonchus infects the liningof the abomasum of ruminants, causing anaemia and weight loss and ifuntreated frequently leads to death. Animals infected with the relatednon-blood feeding nematode Ostertagia similarly fail to thrive and maydie if untreated.

Other parasitic worms of economic importance include the various speciesof the following helminth genera: Trichostrongylus, Nematodirus,Dictyocaulus, Cooperia, Ascaris, Dirofilaria, Trichuris and Strongylus.In addition to domestic livestock, pets and humans may also be infected,not infrequently with fatal results and helminth infections andinfestations thus pose a problem of considerable worldwide significance.

Control of helminth parasites of grazing livestock currently reliesprimarily on the use of anthelmintic drugs combined with pasturemanagement. Such techniques are often unsatisfactory firstly, becauseanthelmintic drugs may have to be administered frequently, secondlybecause resistance against anthelmintic drugs is becoming increasinglywidespread and thirdly because appropriate pasture management is oftennot possible on some farms and even where it is, it can placeconstraints on the best use of available grazing.

Numerous attempts have been made to control helminth parasites ofdomestic animals by immunological means. With very few exceptions (e.g.the cattle lungworm, Dictyocaulus viviparus) this has not provedpossible.

A vaccine against F.hepatica has been proposed in WO90/08819 comprisinga glutathione-S-transferase from F. hepatica as antigenic material.

Bennett (UK Patent No. 2169606B) extracted various antigens fromFasciola organisms by a process which separates antigens specific to thejuvenile stage from antigens present throughout the juvenile and adultstages.

It is known that in vitro cultured F. hepatica release protease enzymeswhich are capable of cleaving immunoglobulins with a papain orCathepsin-B type of activity (Chapman and Mitchell, Vet. Parasitol. 11(1982), p. 165-178). It has been suggested that these protease enzymesmay assist in evading the immune response in combination with the knownability of the worms to slough off the surface glycocalyx thus sheddingbound antibody (Hanna, Exp. Parasitol 50 (1980), p. 155-70). Furthermorecrude in vitro excretory/secretory products can under some circumstancesconfer immunity on rats (Rajasekariah et al, Parasitol. 79 (1979), p.393-400) perhaps by raising antibodies to such enzymes thus inhibitingthem. However, the precise nature of the enzymes is far from clear.

A study of excretory/secretory proteases involving gelatin substratepolyacrylamide gel electrophoresis (GS-PAGE) (Dalton and Heffernan, Mol.Biochem. Parasitol. 35 (1989), p. 161-166) showed a number of cysteineproteases with a wide range of molecular weights and falling generallyinto two groups, namely from 27.5 KDa to 46 KDa active at pH 4.5″8.0 andfrom 60 KDa to 88 KDa active at pH 3.0-4.5. It was suggested that thelatter group might correspond to the immunoglobulin cleaving enzymes ofChapman and Mitchell and that autolysis and/or aggregation of one ormore protease enzymes might be giving the multiple band structure.

Subsequently an HPLC procedure was used and three peaks resolved.Protein from the 15 kDa peak was found to have the ability to cleave IgGat an optimum of pH 4.5 (Heffernan et al, Biochem. Soc. Trans. 19(1991), page 275).

Another study attempting to characterise the protease enzymes of adultF. hepatica is that of Rege et. al. (Mol. Biochem. Parasitol. 35 (1989),p. 89-96) in which a 14,500 Da protein was purified by cation exchangechromatography and molecular sieve HPLC. Maximal hydrolysis of thesubstrate CBZ-Phe-Arg-AFC was found at pH 6.0. Rege et al usedlyophilised whole worms as the source of their protease so that it isnot clear whether their protease is excreted or not. They speculatedthat the protease might be involved in immune evasion or nutrition.

A protease isolated from “Fasciola spp.” has been reported by Yamasakiet al. (Japan J. Parasitol., 38 (1989), p. 373-384). The protease had amolecular weight of 27 kDa as determined by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE), was capable ofhydrolysing haemoglobin and this hydrolysing activity was inhibited bycysteine protease inhibitors. Monoclonal antibodies specific for theprotease could also inhibit the haemoglbin hydrolysis.

Other studies relating to proteases released by helminth parasites areFagbemi and Hillyer, Vet. Parasitol. 40 (1991), p. 217-226 relating toFasciola gigantica; Knox and Kennedy, Mol. Biochem. Parasitol. 28(1988), p. 207-216 relating to Ascaris suum; and Yamakami and Hamajima,Comp. Biochem. Physiol. 87B (1987), p. 643-648 relating to Paragonimuswestermani.

SUMMARY OF THE INVENTION

It has now been found that the group of cysteine proteases disclosed byDalton and Heffernan as having a range of molecular weights in the range27.5-88 KDa on gelatin substrate (GS) PAGE can in fact be resolved astwo proteases of 27 KDa and 29.5 by SDS-PAGE under reducing conditions;that these proteases are two distinct Cathepsin L-like activities asdetermined by substrate specificity, affinity for ion exchange columnsand N-terminal sequencing; that the proteases also have the ability tocleave immunoglobulins; that immunisation of rabbits with purifiedproteases can stimulate antibodies capable of neutralizing the enzymeactivity; and that this discovery opens up the possibility of aneffective vaccine against helminth parasites and in particular F.hepatica using well-characterised purified protective antigens andavoiding the drawbacks in terms of toxicity and side-effects such asimmune suppression or dominance which are inherent in the use ofunresolved crude excretory/secretory products.

Accordingly a first aspect of the present invention provides a vaccinefor use in combating a parasitic infestation of helminths in a mammalwherein the antigenic material comprises a protease having enzymeactivity of the Cathepsin L type, in at least partially purified form,or an antigenic fragment or epitope thereof, together with a carrierand/or adjuvant.

The invention also provides a method of combating a parasiticinfestation of helminths in a mammal comprising administering to saidmammal a vaccine according to the invention as hereinbefore defined inan amount effective to combat said infestation.

The mammal is preferably a ruminant, for example cattle or sheep, butthe vaccine and method of the invention may also find application inhumans.

DETAILED DESCRIPTION OF THE INVENTION

Preferably the Cathepsin L-like protease is derived from flukes such asFasciola or Dicrocoelium, in particular from the liver fluke Fasciolahepatica. Alternatively it is preferred that the Cathepsin L-likeprotease should be capable of stimulating an immune response which willbe effective against Fasciola or Dicrocoelium, in particular F. hepaticaand F. gigantica, such Cathepsin L-like-molecules from other species asare capable of conferring a cross-protective immune response thusforming a particularly preferred aspect of the invention.

The F. hepatica Cathepsin L-like protease shown hereinafter to possess amolecular weight of approximately 27 KDa by sodium dodecyl sulphatepolyacrylamide gel electrophoresis under reducing conditions isparticularly preferred for use in the vaccine and method of theinvention. This protease also gives an apparent molecular weight bymolecular sieve HPLC of 15 KDa. It will be referred to as Cathepsin L1.

The F. hepatica Cathepsin L-like protease shown hereinafter to possess amolecular weight of approximately 29.5 kDa by sodium dodecyl sulphatepolyacrylamide gel electrophoresis under reducing conditions is alsoparticularly preferred for use in the vaccine and as a novel proteinitself forms a further aspect of the invention. This Cathepsin will bereferred to as Cathepsin L2 where necessary to distinguish it fromCathepsin L1.

The Cathepsin L-like activity incorporated in the vaccine according tothe invention is in at least partially purified form. Preferably theCathepsin L-like activity comprises at least 75% of the totalexcretory/secretory proteins present in the vaccine and more preferablythe Cathepsin L is at least 95% pure. It will be appreciated that onceCathepsin L of at least 95% purity has been obtained it can be admixedwith one or more further purified antigenic proteins, including one ormore further excretory/secretory proteins, to form a polyvalent vaccine.

Cathepsin L-like activity can be demonstrated by the ability to cleavethe synthetic peptide substrate Z-phe-arg-AMC(benzyloxycarbonyl-L-phenylalanyl-L-arginyl-7-amido-4-methyl coumarin)combined with a relative inability to cleave the related peptidesZ-arg-arg-AMC and Z-arg-AMC thus distinguishing the enzyme fromCathepsin B and Cathepsin H. Confirmation of the protease as a CathepsinL can also be obtained by a comparison of the N-terminal amino acidsequence with the sequences of known Cathepsin L molecules.

Rat liver Cathepsin L, a mammalian Cathepsin L, has been shown to existas a two chain protein (Ishidoh et al, FEBS Letters 223 (1987), pages69-73). It is unclear whether or not a similar structure is present innon-mammalian Cathepsin L, although see the amino acid sequencingresults presented hereinafter.

The vaccines according to the invention may be formulated withconventional carriers and/or adjuvants and the invention also provides aprocess for the preparation of the vaccines comprising bringing intoassociation a purified protease having enzyme activity of the CathepsinL type or an antigenic fragment or epitope thereof and one or moreadjuvants or carriers. Suitable adjuvants include aluminium hydroxide,saponin (ISCOMs), muramyl dipeptide, mineral and vegetable oils, DEAEdextran, nonionic block copolymers or liposomes such as Novasomes (TradeMark of Micro Vesicular Systems Inc.), in the presence of one or morepharmaceutically acceptable carriers or diluents. Carriers for peptidesequences corresponding to epitopes of Cathepsin L-like proteaseaccording to the invention can be proteins such as Hepatitis B coreantigen multiple antigen peptide or lipopeptides such astripalmitoyl-S-glycerylcysteinylserylserine (P₃CSS). Suitable diluentsinclude liquid media such as saline solution appropriate for use asvehicles. Additional components such as preservatives may be included.

Administration of the vaccine to the host species may be achieved by anyof the conventional routes, e.g. orally or parenterally such as byintramuscular injection, optionally at intervals e.g. two injections ata 7-35 day interval. A suitable dose when administered by injectionmight be such as to give an amount of Cathepsin L-like protein withinthe range 10-500 μg.

While the Cathepsin L-like protease for use in the vaccine according tothe invention may be prepared by isolation from the excretory/secretoryproducts of adult and/or juvenile helminths, it may also be convenientto prepare it by recombinant DNA techniques with the known advantageswhich such techniques give in terms of purity of product, scaling-up ofproduction and reproducibility. Thus the invention also provides aCathepsin L-like protease or a proenzyme therefor or an antigenicfragment or epitope thereof, produced by means of recombinant DNAtechniques.

Additional aspects of the invention related to the above include DNAmolecules encoding for Cathepsin L-like proteases or antigenic fragmentsor epitopes thereof; vectors containing one or more such DNA sequences;host cells, for example bacteria such as E. coli or more preferablyeukaryotic cells, transformed by such vectors, for example by abaculovirus vector; and processes for preparing recombinant CathepsinL-like protease or antigenic fragments or epitopes thereof comprisingculturing such transformed host cells and isolating said CathepsinL-like protease or fragment or epitope from the cultured cells. Sincethe tertiary structure of the Cathepsin L-like protease is important inthe antibody response of a vaccinated animal eukaryotic expressionsystems are preferred as the tertiary structure will be more faithfullyreproduced.

An alternative live or inactivated vaccine formulation may comprise anattenuated or virulent virus or a host cell, e.g. a microorganism suchas a bacterium, having inserted therein a DNA molecule according to theinvention for stimulation of an immune response directed againstpolypeptides encoded by the inserted nucleic acid molecule.

Additional antigenic materials may also be present in the vaccine thusgiving an enhanced protective effect against the helminth parasite inquestion or a combined protective effect against one or more additionalparasitic infestations.

A yet further aspect of the invention provides a monoclonal orpolyclonal antibody capable of inducing immunity to a Cathepsin L-likeprotease in a mammal when administered to said mammal, the antibodyhaving an affinity for the variable region of one or more furtherantibodies, said further antibodies having an affinity for saidCathepsin L.

This approach, the so-called “anti-idiotype” approach, permitsformulation of a vaccine which will dispense entirely with the originalantigen and may offer even greater advantages in terms of safety,avoidance of side effects and convenience of manufacture.

The invention is illustrated by the following examples:

1) CATHEPSIN-L1 PURIFICATION

Mature flukes were obtained from the infected livers of condemnedanimals at an abattoir. The flukes were washed, cultured overnight, andthe culture medium centrifuged as described in Dalton and Heffernan,Mol. Biochem. Parasitol 35 (1989) p. 161-166. 500 ml of the culturemedium (E/S) was concentrated to 10 ml by ultrafiltration with amembrane having a 3000 mw cut off and the sample was applied at 4° C. toa 120 ml Sephacryl S-200 column (1.9×42 cm, Pharmacia, Uppsala, Sweden)equilibrated in phosphate buffered saline (PBS) pH 7.3; the void volumeof the column was found to be 110 ml. Fractions (volume 5 ml) werecollected (after the void volume had been eluted), and they weremonitored for protein content at O.D. 280 nm, using a LKB Uvicordmonitor. The Cathepsin L-like enzyme activity in the different fractionswas assayed using the synthetic fluorogenic peptide Z-Phe-Arg-AMC, knownto be specific for Cathepsin-L enzymes. The release of the fluorescentleaving group, 7-amino-4-methylcoumarin (AMC) was monitored in aPerkin-Elmer Luminescence Spectrometer model LS 50, at excitation andemission wavelengths of 370 and 440 nm, respectively (FIG. 1A).Fractions with Cathepsin L-like activity were pooled and applied to a 50ml QAE Sephadex (Pharmacia, Uppsala, Sweden) column. The column wasprepared as follows:

QAE Sephadex A-50 was preswollen in 0.1M Tris-HCl pH 7.0 supplementedwith 1.0M NaCl and poured into a 50 ml column. The column was thenequilibrated in 0.1M Tris pH 7.0. The pooled cathepsin L-like proteasewas reapplied twice at 4° C. to ensure maximum binding of non-cathepsinL proteins present in the E/S products. The flowrate of the column wasapproximately 1 ml/min. The run through fraction i.e. the fractioncontaining proteins not adsorbed to the QAE Sephadex (approximately 50ml) was collected and concentrated to a volume of 10 ml byultrafiltration with a membrane having a 3000 mw cut off, dialysedagainst ultra-pure water and freeze dried.

The final specific activity of the purified cathepsin-L1 proteinease was11.67×10³ units/mg protein (where one unit releases 1 μmol of AMC/min).

Samples from the purification were taken and loaded onto GS,SDS-non-reducing and SDS-reducing polyacrylamide gels (10% acrylamide,0.1% SDS) in order to evaluate the proteinase activity, and to establishthe purity of the cathepsin-L1. The result is shown in FIG. 1B in whichlanes 1 and 3 are total E/S products on GS and SDS non-reducing gels,lanes 2 and 4 are the purified Cathepsin L1 on GS and SDS non-reducinggels while lane 5 is purified Cathepsin L1 on an SDS gel under reducingconditions. GS-PAGE analysis showed that the purified cathepsin-L1enzyme consisted of multiple bands of proteinase activity in themolecular weight range 60 KDa and higher (FIG. 1B, lane 2, cf. Dalton &Heffernan referred to above), these bands correspond to bands of similarmolecular weight on an SDS non-reducing gel while on the SDS-reducinggel these multiple bands were resolved into one band of molecular weight27 KDa (FIG. 1B, lane 5).

2) N-TERMINAL SEQUENCE DETERMINATION

To confirm the classification of this purified enzyme as a cathepsinL-like proteinase, a concentrated sample of pooled cathepsin-L1 from thegel filtration column was sequenced using an Applied Biosystems 477Aprotein sequencer. The resulting amino-acid sequence was aligned withpreviously determined sequences of known cathepsin-L molecules and thecatheps in-B sequence of Schistosoma mansoni (Table 1 below). Identicalresidues to those in F. hepatica cathepsin-L1 are shown by the dots. Inthe first 19 residues of the N-terminal sequence the fluke cathepsin-Lhas 63% identity with cathepsin-L molecules from both bovine and chickenliver sources, 53, 59 and 53% homology with cathepsin-L molecules fromrat, human and Tryanosoma cruzi respectively, and 26% homology with acathepsin-B from S. mansoni

−1 1 2 3 4 5 6 7 8 9 10 11     12 13 14     15 16 17 18 19 F. hepatica(C-L1)  A V P D K I D P R E  S  G - -  Y  V  T - -  G  V  K  D  Q (Seq.ID No: 1)      . .     .   . .     .         .  .         .  .  .  .     . .     .   . .     .         .  .         .  .  .  . Bovine (C-L)   L P D S V D W R E  K  G - -  G  V  T - -  P  V  K  D  Q (Seq. ID No:2)      .       .   . .     .         .  .         .  .  .  .     .       .   . .     .         .  .         .  .  .  . Chicken liver(C-L)  - A P R S V D W R E  K  G - -  Y  V  T - -  P  V  K  D  Q (Seq.ID No: 3)      .       .   . .     .         .  .         .  .  .  .     .       .   . .     .         .  .         .  .  .  . Rat liver(C-L)  - I P R S V D W R E  K  G - -  Y  V  T - -  P  V  K  D  Q (Seq.ID No: 4)      .       .   . .     .         .  .         .  .  .  .     .       .   . .     .         .  .         .  .  .  . Human liver(C-L)  - A P R S V D W R E  K  G - -  Y  V  T - -  P  V  K  D  Q (Seq.ID No: 5)      .       .   . .     .         .  .         .  .  .  .     .       .   . .     .         .  .         .  .  .  . T. cruzi(c-p)  - A P A A V D W R A  R  G - -  A  V  T - -  A  V  K  D  Q (Seq.ID No: 6)      .       .   .      .       .   . S. mansoni (C-B) - I P S N F D S R K  K  W P G  C  K  S I A  T  I  R  D  Q (Seq. ID No:7)

3) CATHEPSIN-L1 ANTIBODY PRODUCTION

Polyclonal antiserum against cathepsin-L1 was raised in white rabbits bysubcutaneous injection of 52 μg of purified enzyme in complete Freund'sadjuvant. Initial immunization was followed by boosts of 52 μg of enzymein incomplete adjuvant at 40 days, 90 days, 120 days and 150 days; 1week later the animals were bled. The antibody titre was determinedusing ELISA. Microtitre plates were coated with 50 μl of adult E/Sproducts and left uncovered at 37° C. overnight. IgG2a immunoglobulinswere purified from the anti-serum using a protein-A column.

4) WESTERN BLOTTING EXPERIMENTS

Western blot immuno-analysis was used to determine the specificity andconsequently the cross-reactivity of rabbit antibodies raised againstthe purified proteinase cathepsin L1 from F. hepatica. These werecarried out using E/S products and purified cathepsin-L1. Proteins wereseparated by SDS-PAGE and electrophoretically transferred tonitrocellulose paper using a semi-dry blotting system. One percent fetalcalf serum (FCS), and 0.5% Tween-20 in PBS was used to block anynon-specific binding sites. The nitrocellulose was incubated withanti-Cathepsin-L1, and the bound immunoglobulin was detected usingalkaline phosphatase-conjugated anti-rabbit serum. Nitro bluetetrazolium and 5-bromo-5-chloro-3-indolyl phosphate prepared indimethylformamide were used as substrate.

The purified immunoglobulins showed no reaction with other proteinspresent in the E/S products and all of their binding specificity wasconfined to the protein bands (MW 60 KDa and higher on SDS-PAGE undernon-reducing conditions) in the E/S products responsible for theproteinase activity of the purified cathepsin-L1. A blot of SDS-PAGE rununder reducing conditions, probed with the anti-cathepsin-L1 antiserum,showed specific binding with only one band of protein MW 27 KDa in bothE/S and purified cathepsin-L1 which correlates to the single band ofprotein seen when an SDS reducing gel of the purified cathepsin-L1 isrun.

5) INHIBITION OF ENZYME ACTIVITY

To determine whether the anti-cathepsin-L1 antibody raised in the rabbitinhibited the activity of the cathepsin-L1, the purified antibodies wereused as follows. FIG. 2A shows a 10% gelatin substrate non-reducingpolyacrylamide gel, loaded with cathepsin-L1 and increasing amounts ofanti-cathepsin-L1 IgG. It is evident from this gel that with increasingconcentrations of anti-cathepsin-L1 IgG, the proteinase activity of theenzyme decreases as shown by the decreasing intensity of the clear bandson the dark background (lanes 1-7), whereas similar concentrations ofnon-immune rabbit IgG had no effect on the proteinase activity ofcathepsin-L1 (lane 8 cathepsin L alone, lane 9 cathepsin L withnon-immune rabbit IgG).

One of the most striking properties of the Cathepsin-L1 from F. hepaticais its ability to cleave antibody at the hinge region. Purifiedcathepsin-L1 was mixed with anti-cathepsin-L1 IgG or control IgG,incubated at 37° C., samples taken at various time intervals and theantibody molecules in the reaction mixture analysed by SDS-PAGE.Densitometric scans of these gels demonstrate that the binding of theanti-cathepsin-L1 to the enzyme prevents it from gaining access to thecleavage site in the antibody heavy chain; hence, the peaks representingthe fragments generated from digestion of the heavy chain (FIG. 2, panelB, (ii)), are much reduced compared to those observed when cathepsin-L1is incubated with control IgG (FIG. 2, panel B(i)).

6) CHARACTERISATION OF IGG CLEAVING ENZYME IN ADULT FLUKE E/S PRODUCTS

A mouse monoclonal antibody IgG2a obtained by known methods was used asa model substrate. The antibody was incubated with a sample of adult F.hepatica E/S products, or with the thiol proteinase papain. SDS-PAGEanalysis revealed that proteinases in the E/S products cleave the mouseIgG2a heavy chain into two fragments. These fragments were similar inmolecular size to the fragments produced by papain. Therefore, adultflukes secrete an enzyme capable of cleaving IgG2a close to the papaincleaving site, that is, within the hinge region of the antibody heavychain. The fluke enzyme is not specific for IgG2a antibodies but alsocleaved mouse IgG2b and IgG1a purified by protein-A affinitychromatography from whole mouse and rabbit serum.

Mature fluke E/S products were also analysed by HPLC. E/S products wereconcentrated ten-fold by freeze-drying and dialysed overnight against0.1M potassium phosphate, pH 7.0. The dialysate was then filteredthrough a 0.45 μM membrane filter (Gelman Sciences, Michigan, USA) and100 μg samples subjected to molecular sieve HPLC on a TSK3000SW column(Waters, Milford, USA). The mobile phase was 0.1M potassium phosphate,pH 7.0, the flow rate was 0.3 ml/min and the eluted proteins weremonitored by absorbance at 280 nm using a sensitivity range of 0.05. Themolecular sizes of proteins were determined by calibrating the columnwith the following proteins; IgG2a (150 kDa), bovine serum albumin (67kDa), horse radish peroxidase (45 kDa) and lysozyme (14.3 kDa).

Analysis by GS-PAGE of the proteins eluted from HPLC yielded three majorprotein peaks of >150 kDA (Peak I), 45 kDa (Peak II) and 15 kDa (PeakIII), see FIG. 3a. A sample of each fraction was incubated with theIgG2a monoclonal antibody and the products of the reaction subjected toSDS-PAGE. The IgG2a cleaving enzyme was associated with the third, 15kDa, peak, see FIG. 3b.

Each fraction was also tested for-Cathepsin L-like activity using thesynthetic peptide substrate Z-phe-arg-AMC. Cathepsin L-like activity wasassociated with the 15 kDa peak. The optimum pH for Cathepsin L-likeactivity was determined for the 15 kDa protease using Z-phe-arg-AMC, seeFIG. 3C.

GS-PAGE analysis was then carried out on pooled fractions from eachpeak. The proteolytic bands detected in the total adult fluke E/Sproducts using GS-PAGE were present in either the 45 kDa or 15 kDa peak,no proteinases were associated with the >150 kDa peak GS-PAGE analysisof the 45 kDa Peak II showed that it contained several proteinasesranging from 46 to 27.5 kDa; these proteinases correlated exactly withthe Group 2 proteinases described by Dalton and Heffernan [Mol. BiochemParasitol. 35 (1989)] as having optimal activity between pH 4.5-8.0. The15 kDa peak consisted of several proteinases; these enzymes showedsurprisingly high apparent molecular sizes ranging between 60-90 kDa.These proteinases correlated with the Group 1 thiol proteinasesdescribed by Dalton and Heffernan [ibid] that have a pH optimum foractivity at pH 4.5.

Clearly therefore the Cathepsin-L1 molecule demonstrated as having a 27kDa molecular weight by SDS-PAGE under reducing conditions shows variousaberrant molecular weights depending on the technique used, thus on HPLCan abnormally low molecular weight is observed while on GS-PAGEautolysis and aggregation appear to give a series of higher molecularweight bands.

7) CATHEPSIN L-LIKE ACTIVITY IN E/S PRODUCTS OF VARIOUS STAGES OF F.hepatica

F. hepatica metacercariae (Pfizer strain) encysted on cellophane wereremoved with a Pasteur pipette into 2% sodium hypochlorite, vortexed andincubated for 30 minutes at 37° C. This procedure removes the outer cystof the metacercariae. The metacercariae, now only with the innertransparent cyst, were placed into microtitre wells with an automaticpipette using a stereomicroscope and washed in distilled water. Theywere then incubated in a medium prepared by mixing equal volumes of0.05M HCl with a solution containing 1% sodium bicarbonate, 0.8% NaCland 0.2% sodium taurocholate. After 3 hours at 37° C. 70-80% of theflukes were excysted and actively moving. The excysted juveniles wereseparated from the inner cysts using a 20 μl automatic pipette under astereomicroscope.

Mature flukes were removed from the bile ducts of bovine livers obtainedat an abbattoir. Immature parasites were obtained from the liver of maleWistar rats three and five weeks after infection with 20 metacercariae.

Newly excysted juvenile (NEJ), 3 week-old, 5 week-old and mature F.hepatica were maintained in vitro over a period of 3 days. The culturemedium, removed daily, was then assayed for Cathepsin L-like activityusing the fluorogenic substrate Z-phe-arg-AMC. Cathepsin L-like activitywas present in the E/S products from all stages examined. Whilst theCathepsin L-like activity increased on a daily basis in the NEJ E/Sproducts, indicating an increase in its secretion with time, theactivity of this proteinase in the EIS of all other stages decreasedover the same time period. The proteinase activity in the E/S of eachliver fluke stage was compared using three arginine-containingfluorogenic peptide substrates. These substrates were chosen on thebasis of their affinity for lysosomal cathepsin enzymes; cathepsin L(Z-phe-arg-AMC), cathepsin B (Z-arg-arg-AMC) and cathepsin H(Z-arg-AMC). Significant activity was only detected using theZ-phe-arg-AMC substrate and similar results were obtained for all liverfluke stages examined (Table 2 below).

TABLE 2 Specific activity against fluorogenic substrates by E/S productsfrom various liver fluke developmental stages* Substrate NEJ 3 weeks 5weeks Adults Z-Phe-Arg-AMC 25 29 90 1254 Z-Arg-Arg-AMC 0 0 2 21Z-Arg-AMC 1 3 5 63 *values represent means of duplicate results in μmolAMC/min/Mg

8) ANTIBODY-CLEAVING ACTIVITY IN E/S PRODUCTS

As IgG is involved in antibody-dependent cellular cytotoxicity againsthelminth parasites and since adult fluke Cathepsin-L1 cleaves mouseIgG2a [see above], the E/S products from NEJ, 3 week-old, 5 week-old andadult flukes were tested for antibody cleaving activity. E/S productsfrom all F. hepatica stages examined were capable of cleaving thepurified IgG at the hinge region of the heavy chain and therebyreleasing the Fc portion from the antibody binding regions. TheCathepsin-L inhibitor, Z-phe-ala-CHN₂, at a final concentration of 50 μMcompletely inhibited the IgG cleaving activity of the E/S products-fromadult and NEJ flukes. DMSO, at a final concentration of 1%, did notinhibit the IgG cleaving activity.

9) EFFECTS OF E/S PRODUCTS AND CATHEPSIN L1 PROTEINASE ON EOSINOPHILADHERENCE

The possible role of the F. hepatica Cathepsin L1 in preventingantibody-mediated attachment of eosinophils to NEJ was examined using anin vitro assay.

Twenty newly excysted juvenile F. hepatica were dispensed into 100 μl ofrat eosinophil-rich suspension in microtitre wells. According to theexperiment either 100 μl aliquots of immune serum or control serum wereadded with or without E/S products or purified Cathepsin L1. Thecysteine proteinase inhibitor leupeptin was added in some experiments ata final concentration of 5 μg/ml. All dilutions were made in RoswellPark Memorial Institute 1640 with 4% heat inactivated fetal calf serum.At the end of the experiment juvenile flukes were carefully transferredto a microscope slide and examined microscopically at 40× and 100×magnifications. Individual flukes were examined and the number of boundeosinophils counted. Those NEJ with more than 20 cells attached wereconsidered positive.

When rat eosinophil-rich cell populations were added to NEJ in thepresence of immune rat serum (IRS obtained from female Wistar ratsorally infected with 30 F. hepatica metacercariae; blood was taken after5 weeks of infection; the sera obtained were pooled, aliquoted andstored at −20° C. until required; control serum was collected fromuninfected rats), cell adherence was consistently high (>90% positiveNEJ after 2 hours, FIG. 4, panel A; the scale along the base of theFigure represents percentage of positive NEJ as defined above; the 5bars represent counts at 2 hour intervals). This cellular adherence wasnot observed in the presence of normal serum (FIG. 4, panel B) and henceit must be presumed that it is mediated by anti-NEJ antibodies. Themaximum cell attachment was registered after 2 hours of incubation.Addition of NEJ or mature fluke E/S products, together with immuneserum, resulted in a >70% reduction in the number of positive NEJ ascompared to immune serum alone (FIG. 4, panels C and D). Similarly,purified Cathepsin L1 prevented the attachment of eosinophils (FIG. 4,panel E). When the cysteine proteinase inhibitor, leupeptin, was addedin the presence of immune serum and either NEJ or mature fluke E/Sproducts or purified Cathepsin L1, eosinophil attachment was similar tothat obtained using immune serum alone (FIG. 4, panels A, F, G and H);hence leupeptin inhibits the effect of the E/S products and CathepsinL1. Leupeptin added to NEJ in culture did not affect their viability(data not shown) although in the presence of immune serum it slightlyreduces the rate at which eosinophils are lost from the NEJ surface(FIG. 4, panel I). When purified Cathepsin L1 was added to the assay 2hours after the addition of immune serum, thereby allowing eosinophilsinitially to attach, these attached eosinophils were quickly detachedfrom the NEJ surface (FIG. 4, panel J, compare panels A and E).

10) CATTLE TRIALS

Eighteen cattle were housed indoors. The cattle were allocated into 5groups, A, B, C, D and E, each of three or four cattle and acclimatisedfor a period of 7 days. Primary immunisation occurred on day 0. Animalsin group A were negative controls and received 150 μg of horse spleenferritin, groups B, C, D and E received 10, 50, 200 and 500 μg ofCathepsin L1 respectively prepared as described under (1) above, all byinjection. Primary immunisations were adjuvanted in Freund's completeadjuvant (FCA). Twenty-eight days later all animals received a secondaryimmunisation and also a tertiary vaccination at day 56. Thesevaccinations were adjuvanted in Freund's incomplete adjuvant (FIA).Antibody titres were monitored throughout this period. On day 76 allanimals received 25 ml levamisole after nematode eggs were found infaecal samples from some of them.

On day 84 all groups of cattle received an exogenous challenge of ca 500liver fluke metacercariae administered by gelatin capsule from a dosinggun (F. hepatica was of UK origin). The progress of the infection wasmonitored via levels of enzymes in the blood and faecal egg counts.

Summary of animal groups: Number of Group ID Animals Vaccination A 4 150μg horse spleen ferritin B 4  10 μg Cathepsin L1 C 4  50 μg Cathepsin L1D 3 200 μg Cathepsin L1 E 3 500 μg Cathepsin L1

All animals responded to immunisation as determined by ELISA—followingchallenge all animals showed increased serum levels of glutamicdehydrogenase and glutamyl gamma transferase, indicative respectively ofliver parenchyma and bile duct damage caused by the liver flukes. Onlyanimals in the control group showed eggs in faeces.

11) CATHEPSIN-L2 PURIFICATION

Flukes were washed, cultured and the culture medium centrifuged asdescribed above for Cathepsin L1. Five hundred ml of the E/S productswere thawed, concentrated to a volume of 10 ml on an Amicon 8400concentrator (Amicon, Wis., USA) using an Amicon YM3 membrane (3,000 Dam.w. cut-off), and applied to a gel filtration column containingSephacryl S200HR resin (1.9 cm×42 cm, Pharmacia, Uppsala, Sweden)equilibrated in 0.1M tris-HCl pH 7 at 4° C. The column was eluted with0.1M Tris-HCl pH 7 and 70×5 ml fractions were collected. The fractionscontaining cathepsin L2 activity, assayed, using the fluorogenicsubstrate Tos-Gly-Pro-Arg-AMC in 0.1M glycine at pH 7, on a Perkin-ElmerFluorescence spectrophotometer with an excitation wavelength of 370 nmand an emission filter setting of 440 nm, were pooled.

The Sephacryl S200 fraction was applied to a 50 ml QAE Sephadex column(2.5 cm×10.0 cm, Pharmacia, Uppsala, Sweden) equilibrated in 0.1Mtris-HCl pH 7. The QAE Sephadex column was washed with 300 ml of 0.1MTris-HCl pH 7 and 150 ml of 75 mM NaCl in 0.1M Tris-HCl pH7 and wassubsequently eluted with 250 ml of 0.4M NaCl in 0.1M Tris-HCl pH 7. Fiveml fractions from each washing and elution step (180 fractions in total)were collected. The fractions found to contain Tos-Gly-Pro-Arg-AMCcleaving activity were pooled (QAE400 fraction). The QAE400 fraction wasconcentrated to 20 ml on a Amicon 8400 concentrator using a YM3 membraneand then diluted with distilled water to a volume of 100 ml. The dilutedQAE400 fraction was then concentrated to a final volume of 10 mlcontaining a NaCl concentration calculated to be approximately 80 mM.The concentrated QAE400 fraction was stored as 10×1 ml aliquots at −80°C.

The homogeneity of the purified cathepsin L2 was determined usingdenaturing SDS-PAGE gels containing 12% polyacrylamide. The purifiedcathepsin L2 migrates as a single band of 29.5 kDa on a reducingSDS-PAGE gel (FIG. 5C, lane 1 molecular weight markers, lane 2 adultfluke E/S products, lane 3 purified Cathepsin L2).

Zymography was performed using 12% PAGE gels according to the method ofDalton and Heffernan, Mol. Biochem. Parasitol. 35 (1989) p. 161-166,both in the presence and absence of SDS. Analysis in the presence of SDSshows that the enzyme migrates as 4 bands which are also observed in thetotal adult fluke E/S products (FIG. 5A, lane 1 adult fluke E/Sproducts, lane 2 purified cathepsin L2). However, zymography in theabsence of SDS shows that the purified cathepsin L2 migrates as a singleproteolytic band (FIG. 5B, lanes as in FIG. 5A).

12) AMINO-TERMINAL SEQUENCE ANALYSIS

A 5.4 ml aliquot of E/S products (5 mg protein approx.) was concentratedto 200 μl by freeze-drying. Forty μl of concentrated sample was appliedto a non-denaturing 12% SDS-PAGE gel and electrophoresed as describedabove. After electrophoresis the gel was incubated for 30 minutes intransfer buffer (25 mM Tris, 190 mM glycine and 10% (v/v) methanol). Astrip of PVDF (Problott) membrane (10 cm×4.5 cm) was immersed inmethanol for 10 sec followed by equilibration in transfer buffer for 5minutes. Separated proteins were transfered to the PVDF membrane using asemi-dry electroblotting apparatus (Atto corp., Tokyo, Japan) accordingto the manufacturers instructions. The membrane was stained anddestained and air-dried and the protein bands of interest were cut outand sequenced on an Applied Biosystems 477A protein sequencer at theUniversity of Cambridge.

The N-terminal sequence is given below in Table 3 aligned withN-terminal sequences determined for F. hepatica cathepsin L1, cathepsinL from chicken, rat and human livers, cathepsin S from bovine spleen, aCathepsin L-like protease from T.cruzi (cruzipain) and cathepsin B fromS. mansoni. It can be seen cathepsin L1 is 93% homologous to cathepsinL2 (one amino acid, arginine in position 7 in cathepsin L2 issubstituted for a proline in position 7 in cathepsin L1). Cathepsin L2is only 47% homologous to chicken liver cathepsin L, rat liver cathepsinL and human liver cathepsin L, 47% homologous to bovine spleen cathepsinS, 40% homologous to the T. cruzi cathepsin L-like protease (cruzipain)and 20% homologous to S. mansoni cathepsin B.

By contrast with the two chain structure known to exist for mammalianCathepsin L, the finding of a single N-terminal amino acid sequence forboth Cathepsin L1 and L2 implies the presence of only a single chain. Itis however possible that a second N-terminally blocked chain is present.

−1 1 2 3 4 5 6 7 8 9 10 11     12 13 14 F. hepatica (cathepsin L2) A V P D K I D R R E  S  G - -  Y  V (Seq. ID No: 8) F. hepatica(cathepsin L1)  A V P D K I D P R E  S  G - -  Y  V  T (Seq. ID No: 9)Chicken liver (cathepsin L)  - A P R S V D W R E  K  G - -  Y  V  T(Seq. ID No: 11) Rat liver (cathepsin L) - I P R S V D W R E  K  G - -  Y  V  T (Seq. ID No: 11) Human liver(cathepsin L)  - A P R S V D W R E  K  G - -  Y  V  T (Seq. ID No: 12)Bovine spleen (cathepsin S)    L P D S M D W R E  K  G - -  C  V  T(Seq. ID No: 13) T. cruzi (c-p)  - A P A A V D W R A  R  G - -  A  V  T(Seq. ID No: 14) S. mansoni (C-B) - I P S N F D S R K  K  W P G  C  K  S (Seq. ID No: 15)

13) KINETIC STUDIES

The kinetic constants of F. hepatica cathepsins L1 and L2 enzymes weredetermined for 11 different substrates: Z-Phe-Arg-AMC,Bz-Phe-Val-Arg-AMC, Suc-Leu-Leu-Val-Tyr-AMC, H-Leu-Val-Tyr-AMC,Tos-Gly-Pro-Lys-AMC, Tos-Gly-Pro-Arg-AMC, Boc-Val-Pro-Arg-AMC,Z-Arg-Arg-AMC, Z-Arg-AMC, Suc-Ala-Phe-Lys-AMC and Boc-Val-Leu-Lys-AMC.One mg of fluorogenic substrate was dissolved in 100 μl of dimethylformamide. This stock solution was diluted in 0.1M glycine pH 7.0 toachieve the desired concentration of substrate. Each substrateconcentration was in triplicate and the final assay volume was 1.0 ml.Included in the 1 ml aliquot was 20 μl of enzyme and 50 μl of 10 mMdithiothreitol. The samples were incubated at 37° C. for 30 min beforestopping the reaction with the addition of 200 μl of 1.7 M acetic acid.The samples were assayed for released 7-amino-methyl-coumarin as above.The kinetic constants V_(max) and K_(m) were obtained by non-linearregression according to the method of Barrett et al (Biochem J. 201,p.189-198) except that 20 μl of cathepsin L2 was incubated with 20 μl of1.0 μM-0.1 μM E-64 in a final volume of 80 μl 0.1M glycine pH 7.0 for 30mins at 37° C. Twenty μl of 1/10 cathepsin L1 was incubated with 20 μlof 5 μM-0.5 μM E-64 in a final volume of 80 μl 0.1M glycine pH 7.0 for30 min. at 37° C. All of the incubated sample was assayed for thefluorogenic substrate Z-Phe-Arg-AMC as above.

The kinetic constant results are shown in Table 4. The data show thatboth enzymes favour the substrate Boc-Val-Leu-Lys-AMC with cathepsin L2having 2.5 times the affinity (k_(cat)/K_(m)) for Boc-Val-Leu-Lys-AMC ofcathepsin L1. The second best substrate was Z-Phe-Arg-AMC with cathepsinL2 having 2 times higher affinity for this substrate than cathepsin L1.Cathepsin L2 cleaves the fluorogenic substrates H-Leu-Val-Tyr-AMC,Bz-Phe-Val-Arg-AMC, Tos-Gly-Pro-Lys-AMC, Tos-Gly-Pro-Arg-AMC andBoc-Val-Pro-Arg-AMC with similar affinities (k_(cat)/K_(m)=38-11310⁻³M⁻¹.S⁻¹). Cathepsin L1 does not cleave these substrates to anysignificant degree (k_(cat)/K_(m)=0.26-3.89 10⁻³ M⁻¹.S⁻¹).

TABLE 4 Enzyme kinetic studies of F. hepatica cathepsins L1 and L2.K_(M) K_(cat) K_(cat) /K_(m) Enzyme Sustrate     (μM) (s⁻¹) (10³s⁻¹ ·M⁻¹) Cathepsin L2 z-Arg-AMC 14.08 0.09 6.39 z-Arg-Arg-AMC 9.07 0.02 2.21z-Phe-Arg-AMC 3.76 0.56 148.9 Bz-Phe-Val-Arg-AMC 1.20 0.06 45.83H-Leu-Val-Tyr-AMC 3.75 0.21 55.47 Suc-Leu-Leu-Val-Tyr-AMC 23.33 0.083.25 Boc-Val-Pro-Arg-AMC 24.81 1.19 47.96 Tos-Gly-Pro-Arg-AMC 17.06 1.93113.3 Tos-Gly-Pro-Lys-AMC 35.49 1.37 38.60 Suc-Ala-Phe-Lys-AMC 41.150.14 3.40 Boc-Val-Leu-Lys-AMC 7.34 3.75 510.9 Cathepsin L1 z-Arg-AMC20.36 0.04 2.16 z-Arg-Arg-AMC 65.60 0.002 0.03 z-Phe-Arg-AMC 14.68 1.0873.57 Bz-Phe-Val-Arg-AMC 9.25 0.03 3.03 H-Leu-Val-Tyr-AMC 5.40 0.02 3.89Suc-Leu-Leu-Val-Tyr-AMC 38.48 0.01 0.34 Boc-Val-Pro-Arg-AMC 43.60 0.020.46 Tos-Gly-Pro-Arg-AMC 26.04 0.03 1.23 Tos-Gly-Pro-Lys-AMC 106.9 0.030.26 Suc-Ala-Phe-Lys-AMC 65.32 0.05 0.77 Boc-Val-Leu-Lys-AMC 34.70 7.90227.7

14) F.hepatica CATHEPSIN L DNA SEQUENCES, CATHEPSIN L GENES IN OTHERHELMINTH PARASITES

DNA sequences were obtained by amplification of F.hepatica cDNA usingconventional polymerase chain reaction (PCR) techniques. The sequencesare shown in FIGS. 6-8 (Seq. ID No: 16-21) with the amino acids forwhich they code. Genomic DNA from other helminth parasites was probedwith the F.hepatica sequence JDCLONEC shown in FIG. 6 using the Southernblotting technique and conditions of moderate stringency. Bands wereobserved in the heartworm (Dirofilaria immitis) and blowfly (Luciliacuprina) channels indicating respectively strong and weak hybridisation.

FIG. 9 is an autoradiograph of one such experiment in which column “A”is λ Hind markers, columns B, C and D are genomic DNA from F.hepatica,Lucilia cuprina and Dirofilaria immitis; Columns E, F and G are bovine,ovine and canine genomic DNA; and finally Column H is λ Hind markersagain.

22 1 20 PRT Fasciola Hepatica 1 Ala Val Pro Asp Lys Ile Asp Pro Arg GluSer Gly Tyr Val Thr Gly 1 5 10 15 Val Lys Asp Gln 20 2 19 PRT Bovine 2Leu Pro Asp Ser Val Asp Trp Arg Glu Lys Gly Gly Val Thr Pro Val 1 5 1015 Lys Asp Gln 3 19 PRT Chicken 3 Ala Pro Arg Ser Val Asp Trp Arg GluLys Gly Tyr Val Thr Pro Val 1 5 10 15 Lys Asp Gln 4 19 PRT Rat 4 Ile ProArg Ser Val Asp Trp Arg Glu Lys Gly Tyr Val Thr Pro Val 1 5 10 15 LysAsp Gln 5 19 PRT Human 5 Ala Pro Arg Ser Val Asp Trp Arg Glu Lys Gly TyrVal Thr Pro Val 1 5 10 15 Lys Asp Gln 6 19 PRT Trypanosoma cruzi 6 AlaPro Ala Ala Val Asp Trp Arg Ala Arg Gly Ala Val Thr Ala Val 1 5 10 15Lys Asp Gln 7 23 PRT S. mansoni 7 Ile Pro Ser Asn Phe Asp Ser Arg LysLys Trp Pro Gly Cys Lys Ser 1 5 10 15 Ile Ala Thr Ile Arg Asp Gln 20 814 PRT Fasciola Hepatica 8 Ala Val Pro Asp Lys Ile Asp Arg Arg Glu SerGly Tyr Val 1 5 10 9 15 PRT Fasciola Hepatica 9 Ala Val Pro Asp Lys IleAsp Pro Arg Glu Ser Gly Tyr Val Thr 1 5 10 15 10 14 PRT Chicken 10 AlaPro Arg Ser Val Asp Trp Arg Glu Lys Gly Tyr Val Thr 1 5 10 11 14 PRT Rat11 Ile Pro Arg Ser Val Asp Trp Arg Glu Lys Gly Tyr Val Thr 1 5 10 12 14PRT Human 12 Ala Pro Arg Ser Val Asp Trp Arg Glu Lys Gly Tyr Val Thr 1 510 13 14 PRT Bovine 13 Leu Pro Asp Ser Met Asp Trp Arg Glu Lys Gly CysVal Thr 1 5 10 14 14 PRT Trypanosoma cruzi 14 Ala Pro Ala Ala Val AspTrp Arg Ala Arg Gly Ala Val Thr 1 5 10 15 16 PRT S. mansoni 15 Ile ProSer Asn Phe Asp Ser Arg Lys Lys Trp Pro Gly Cys Lys Ser 1 5 10 15 16 476DNA Fasciola Hepatica misc_feature (14)..(474) n = unknown 16 t cag ggaaac tgt ngn ncc tgt tgg gca ttc tca aca acc ggt act atg 49 Gln Gly AsnCys Xaa Xaa Cys Trp Ala Phe Ser Thr Thr Gly Thr Met 1 5 10 15 gag ggacaa tat atg aaa aac gaa aaa act agt att tca ttc tct gag 97 Glu Gly GlnTyr Met Lys Asn Glu Lys Thr Ser Ile Ser Phe Ser Glu 20 25 30 caa caa ctggtc gat tgt agc ggt cct tgg gga aat aat ggt tgc agt 145 Gln Gln Leu ValAsp Cys Ser Gly Pro Trp Gly Asn Asn Gly Cys Ser 35 40 45 ggt gga ttg atggaa aat gct tac caa tat ttg aaa caa ttt gga ttg 193 Gly Gly Leu Met GluAsn Ala Tyr Gln Tyr Leu Lys Gln Phe Gly Leu 50 55 60 gaa acc gaa tcc tcttat ccg tac acg gct gtg gaa ggt cag tgt cga 241 Glu Thr Glu Ser Ser TyrPro Tyr Thr Ala Val Glu Gly Gln Cys Arg 65 70 75 80 tac aat agg cag ttggga gtt gcc aaa gtg acc ggc tac tat act gtg 289 Tyr Asn Arg Gln Leu GlyVal Ala Lys Val Thr Gly Tyr Tyr Thr Val 85 90 95 cat tct ggc agt gag gtagaa ttg aaa aat cta gtc ggt tcc gaa gga 337 His Ser Gly Ser Glu Val GluLeu Lys Asn Leu Val Gly Ser Glu Gly 100 105 110 cct gcc gcg atc gct gtggat gtg gaa tct gac ttc atg atg tac agg 385 Pro Ala Ala Ile Ala Val AspVal Glu Ser Asp Phe Met Met Tyr Arg 115 120 125 agt ggt att tat cag agccaa act tgt tta ccg ttc gct ctg aat cat 433 Ser Gly Ile Tyr Gln Ser GlnThr Cys Leu Pro Phe Ala Leu Asn His 130 135 140 gca gtc ttg tct gtc ggttat gga aca cag gat ggt act gntt 476 Ala Val Leu Ser Val Gly Tyr Gly ThrGln Asp Gly Thr 145 150 155 17 157 PRT Fasciola Hepatica misc_feature(5)..(6) Xaa = unknown 17 Gln Gly Asn Cys Xaa Xaa Cys Trp Ala Phe SerThr Thr Gly Thr Met 1 5 10 15 Glu Gly Gln Tyr Met Lys Asn Glu Lys ThrSer Ile Ser Phe Ser Glu 20 25 30 Gln Gln Leu Val Asp Cys Ser Gly Pro TrpGly Asn Asn Gly Cys Ser 35 40 45 Gly Gly Leu Met Glu Asn Ala Tyr Gln TyrLeu Lys Gln Phe Gly Leu 50 55 60 Glu Thr Glu Ser Ser Tyr Pro Tyr Thr AlaVal Glu Gly Gln Cys Arg 65 70 75 80 Tyr Asn Arg Gln Leu Gly Val Ala LysVal Thr Gly Tyr Tyr Thr Val 85 90 95 His Ser Gly Ser Glu Val Glu Leu LysAsn Leu Val Gly Ser Glu Gly 100 105 110 Pro Ala Ala Ile Ala Val Asp ValGlu Ser Asp Phe Met Met Tyr Arg 115 120 125 Ser Gly Ile Tyr Gln Ser GlnThr Cys Leu Pro Phe Ala Leu Asn His 130 135 140 Ala Val Leu Ser Val GlyTyr Gly Thr Gln Asp Gly Thr 145 150 155 18 478 DNA Fasciola Hepaticamisc_feature (15)..(477) n = unknown 18 c cat caa gaa gcc cnn ggc tcttgt tgg gnt ttc tca aca aca ggt gct 49 His Gln Glu Ala Xaa Gly Ser CysTrp Xaa Phe Ser Thr Thr Gly Ala 1 5 10 15 atg gaa gga cag tat atg aaaaac caa aga act agt att tca tnc tct 97 Met Glu Gly Gln Tyr Met Lys AsnGln Arg Thr Ser Ile Ser Xaa Ser 20 25 30 gag caa caa ctg gtc gat tgt agccgt gat ttt ggc aat tat ggt tgt 145 Glu Gln Gln Leu Val Asp Cys Ser ArgAsp Phe Gly Asn Tyr Gly Cys 35 40 45 aat ggt gga cta atg gaa aat gca tacgaa tat ttg aaa cga ttt gga 193 Asn Gly Gly Leu Met Glu Asn Ala Tyr GluTyr Leu Lys Arg Phe Gly 50 55 60 ttg gaa acc gag tct tct tat cct tac agggct gtg gaa gga caa tgt 241 Leu Glu Thr Glu Ser Ser Tyr Pro Tyr Arg AlaVal Glu Gly Gln Cys 65 70 75 80 cga tac aac gag cag ttg gga gtt gcc aaagtg act agc tac tat acg 289 Arg Tyr Asn Glu Gln Leu Gly Val Ala Lys ValThr Ser Tyr Tyr Thr 85 90 95 gta cat tct gga gat gag gta gaa ttg caa aatcta gtc ggt gcc gaa 337 Val His Ser Gly Asp Glu Val Glu Leu Gln Asn LeuVal Gly Ala Glu 100 105 110 gga cct gct gcg gtc gct ttg gat gtg gag tcagac ttc atg atg tac 385 Gly Pro Ala Ala Val Ala Leu Asp Val Glu Ser AspPhe Met Met Tyr 115 120 125 agg agt ggt att tat cag agc caa act tgt tcaccg gat cgt ttg aac 433 Arg Ser Gly Ile Tyr Gln Ser Gln Thr Cys Ser ProAsp Arg Leu Asn 130 135 140 cat gga gtg ttg nct gtc gnt tat gga acn cagggt ggt nctcnc 478 His Gly Val Leu Xaa Val Xaa Tyr Gly Xaa Gln Gly Gly145 150 155 19 157 PRT Fasciola Hepatica misc_feature (5)..(154) Xaa =unknown 19 His Gln Glu Ala Xaa Gly Ser Cys Trp Xaa Phe Ser Thr Thr GlyAla 1 5 10 15 Met Glu Gly Gln Tyr Met Lys Asn Gln Arg Thr Ser Ile SerXaa Ser 20 25 30 Glu Gln Gln Leu Val Asp Cys Ser Arg Asp Phe Gly Asn TyrGly Cys 35 40 45 Asn Gly Gly Leu Met Glu Asn Ala Tyr Glu Tyr Leu Lys ArgPhe Gly 50 55 60 Leu Glu Thr Glu Ser Ser Tyr Pro Tyr Arg Ala Val Glu GlyGln Cys 65 70 75 80 Arg Tyr Asn Glu Gln Leu Gly Val Ala Lys Val Thr SerTyr Tyr Thr 85 90 95 Val His Ser Gly Asp Glu Val Glu Leu Gln Asn Leu ValGly Ala Glu 100 105 110 Gly Pro Ala Ala Val Ala Leu Asp Val Glu Ser AspPhe Met Met Tyr 115 120 125 Arg Ser Gly Ile Tyr Gln Ser Gln Thr Cys SerPro Asp Arg Leu Asn 130 135 140 His Gly Val Leu Xaa Val Xaa Tyr Gly XaaGln Gly Gly 145 150 155 20 473 DNA Fasciola Hepatica misc_feature(1)..(470) n = unknown 20 n gcg aaa tgt ggt tcc tgt tgg gca ttc tca acaacc ggt act atg gag 49 Ala Lys Cys Gly Ser Cys Trp Ala Phe Ser Thr ThrGly Thr Met Glu 1 5 10 15 gga caa tat atg aaa aac gaa aaa act agt ntttca ncc tct gag caa 97 Gly Gln Tyr Met Lys Asn Glu Lys Thr Ser Xaa SerXaa Ser Glu Gln 20 25 30 caa ctg gtc gat tgt agc ggt cct tgg gga aat aatggt tgc agt ggt 145 Gln Leu Val Asp Cys Ser Gly Pro Trp Gly Asn Asn GlyCys Ser Gly 35 40 45 gga ttg atg gaa aat gct tac caa tat tta aaa caa tttgga ttg gaa 193 Gly Leu Met Glu Asn Ala Tyr Gln Tyr Leu Lys Gln Phe GlyLeu Glu 50 55 60 acc gaa tcc tct tat ccg tac acg gct gtg gaa ggt cag tgtcga tac 241 Thr Glu Ser Ser Tyr Pro Tyr Thr Ala Val Glu Gly Gln Cys ArgTyr 65 70 75 80 aat agg cag ttg gga gtt gcc aaa gtg act ggc tac tat actgtg cat 289 Asn Arg Gln Leu Gly Val Ala Lys Val Thr Gly Tyr Tyr Thr ValHis 85 90 95 tct ggc agt gag gca gga ttg aaa aat cta gtc ggt tcc gaa ggacct 337 Ser Gly Ser Glu Ala Gly Leu Lys Asn Leu Val Gly Ser Glu Gly Pro100 105 110 gcc gcg atc gct gtg gat gtg gaa tct gac ttc atg atg tac aggagt 385 Ala Ala Ile Ala Val Asp Val Glu Ser Asp Phe Met Met Tyr Arg Ser115 120 125 ggt att tat cag anc caa act tgt tta ccg ttc gct ttg aat catgca 433 Gly Ile Tyr Gln Xaa Gln Thr Cys Leu Pro Phe Ala Leu Asn His Ala130 135 140 gtc ttg nct gtc gat tat gga aca cag gat ggt nacnccc 473 ValLeu Xaa Val Asp Tyr Gly Thr Gln Asp Gly 145 150 155 21 155 PRT FasciolaHepatica misc_feature (27)..(147) Xaa = unknown 21 Ala Lys Cys Gly SerCys Trp Ala Phe Ser Thr Thr Gly Thr Met Glu 1 5 10 15 Gly Gln Tyr MetLys Asn Glu Lys Thr Ser Xaa Ser Xaa Ser Glu Gln 20 25 30 Gln Leu Val AspCys Ser Gly Pro Trp Gly Asn Asn Gly Cys Ser Gly 35 40 45 Gly Leu Met GluAsn Ala Tyr Gln Tyr Leu Lys Gln Phe Gly Leu Glu 50 55 60 Thr Glu Ser SerTyr Pro Tyr Thr Ala Val Glu Gly Gln Cys Arg Tyr 65 70 75 80 Asn Arg GlnLeu Gly Val Ala Lys Val Thr Gly Tyr Tyr Thr Val His 85 90 95 Ser Gly SerGlu Ala Gly Leu Lys Asn Leu Val Gly Ser Glu Gly Pro 100 105 110 Ala AlaIle Ala Val Asp Val Glu Ser Asp Phe Met Met Tyr Arg Ser 115 120 125 GlyIle Tyr Gln Xaa Gln Thr Cys Leu Pro Phe Ala Leu Asn His Ala 130 135 140Val Leu Xaa Val Asp Tyr Gly Thr Gln Asp Gly 145 150 155 22 12 PRTFasciola Hepatica 22 Ala Val Pro Asp Lys Ile Asp Pro Arg Glu Ser Gly 1 510

What is claimed is:
 1. A vaccine for use in combating a parasiticinfestation of helminths in a mammal, comprising antigenic material,wherein the antigenic material comprises Fasciola hepatica Cathepsin L1having a molecular weight of 27 kDa by sodium dodecyl sulphatepolyacrylamide gel electrophoresis under reducing conditions and beingat least 95% pure, or an antigenic fragment or epitope thereof, togetherwith an adjuvant and, optionally, a carrier.
 2. A vaccine as claimed inclaim 1 wherein the antigenic material is Cathepsin L1 having amolecular weight of 27 kDa by sodium dodecyl sulphate polyacrylamide gelelectrophoresis under reducing conditions and an N-terminal sequenceAVPDKIDPRESG[SEQ ID NO:22].
 3. A method of combating a parasiticinfestation of helminths in a mammal comprising administering to saidmammal a vaccine as claimed in claim 1 in an amount effective to combatsaid infestation.
 4. A method as claimed in claim 3 wherein saideffective amount is within the range 10-500 μg.
 5. A vaccine as claimedin claim 1 further comprising one or more purified antigenic proteins,wherein said proteins be excretory/secretory proteins.
 6. A method asclaimed in claim 3, wherein said antigenic material is Cathepsin L1having a molecular weight of 27 kDa by sodium dodecyl sulphatepolyacrylamide gel electrophoresis under reducing conditions and anN-terminal sequence AVPDKIDPRBSG[SEQ ID NO:22].